Amylin, a 37-amino acid polypeptide structurally related to calcitonin gene-related protein [Cooper, Endocrine Review, 15:163 (1994)], is primarily synthesized, packaged, and secreted from the xcex2-cells of pancreatic islets. Apart from the islet of Langerhans, amylin-like immunoreactivity activity has also been detected in lung, gastrointestinal tract, and the nervous system. See Miyazato, M., et. al., Bioch. Bioph. Res. Comm., 181:293 (1991); Chance, et al., Brain Res., 539:352 (1991); Mulder, et al., Gastroenterology, 107:712 (1994).
The presence of an abnormally high concentration of amylin in the blood, i.e., hyperamylinemia, has been found in patients with pancreatic cancer [Permert, et al., N. Engl. J. Med, 330:313 (1994)], obese patients [Huang, et al., Hypertension, 19 (Supp. I):101 (1992)], and in prediabetic patients [Erickson, J., Diabetologia, 35:291 (1992)]. The hyperamylinemic state has been associated with both diabetes as well as amyloid formations. DeKoning, et al., Proc. Natl. Acad. Sci. USA, 91:8467 (1994). Amyloid formation causes destruction of xcex2-islet cells and eventually pancreatic dysfunction. Johnson, et al., Lab. Invest. 66:522 (1992); Lorenzo, et al., Nature, 368:756 (1994).
Native somatostatin is comprised of both a 14-amino acid isoform (somatostatin-14) and a 18-amino acid isoform (somatostatin-28). Reichlin, New Eng. J. Medicine, 309(24):1495 (1983). Five distinct somatostatin receptors have been identified and characterized. Hoyer, et al., Naunyn-Schmiedeberg""s Arch. Pharmacol., 350:441 (1994). Somatostatin produces a variety of effects, including modulation of hormone release, e.g., growth hormone, glucagon, insulin, amylin, and neurotransmitter release. Some of these effects have been associated with its binding to a specific somatostatin receptor. For example, the inhibition of growth hormone has been attributed to somatostatin type-2 receptor (xe2x80x9cSSTR-2xe2x80x9d). Raynor, et al., Molecular Pharmacol. 43:838 (1993); Lloyd, et al. Am. J. Physiol. 268:G102 (1995). Because of the short half-life of the native somatostatin, various somatostatin analogs have been developed, e.g., for the treatment of acromegaly. Raynor, et al., Molecular Pharmacol. 43:838 (1993).
The present invention is based on the discovery that ligands selective for somatostatin type-5 receptor (xe2x80x9cSSTR-5xe2x80x9d) are effective in inhibiting release of amylin from pancreas cells.
An aspect of this invention relates to a method of determining the ability of a compound to both bind to somatostatin type-5 receptor and inhibit amylin release from amylin-secreting pancreas cells. The method includes the steps of (i) obtaining a preparation, either a cell preparation or a membrane preparation, which contains SSTR-5; (ii) incubating the preparation, the compound, and a SSTR-5 ligand, at least one of the ligand and the compound being detectably labeled; (iii) determining the ability of the compound to compete against the ligand for binding to SSTR-5; (iv) if and only if the compound is determined to be able to bind to SSTR-5, obtaining amylin-secreting pancreatic cells (e.g., cells in an intact pancreas from a rodent such as a rat or a mouse, pancreatic islet cells such as xcex2 cells, or amylinoma cells); (v) incubating the compound, the pancreatic cells, and an amylin release stimulator (e.g., glucose or D-glyceraldehyde) under conditions in which the amylin release stimulator would induce release of amylin from the pancreatic cells; and (vi) determining the ability of the compound to inhibit amylin release.
By xe2x80x9cSSTR-5 ligandxe2x80x9d is meant a compound which binds to SSTR-5, e.g., somatostatin-14, somatostatin-28, an analog of somatostatin-14 or somatostatin-28 (such as [Tyr11]-somatostatin-14), or an antibody raised against of SSTR-5. Either the ligand or the test compound can be labeled with a radioactive isotope, or a nonradioactive (e.g., fluorescent, chemiluminescent, or biotinylated) molecule. Preferably, [125I-Tyr11]-somatostatin-14 is used as a labeled SSTR-5 ligand to practice the above-described method. Other examples of labeled ligands include 125I-LTT-somatostatin-28 [Patel, et al., Endocrinol., 135(6):2814 (1994)] and 125I-CGP 23996 [Raynor, et al., Mol. Pharm. 44:385-392 (1993)].
Examples of the pancreatic cells which are used to practice the above method include both rodent and human islet cells (e.g., xcex2 and xcex4 cells) and pancreatic tumor cells (e.g., amylinoma cells). The pancreatic cells can be incubated either in vitro or in vivo. Examples of an in vitro system include an isolated rat pancreas, the rat xcex2-cell line RINm5f, and the hamster xcex2-cell line H1T-T15. As an example of an in vivo system, Sprague-Dawley or Zucker fatty rats can be used as animal models to test the amylin inhibition activity of test somatostatin analogs. An amylin release stimulator, e.g. 16.7 mm of glucose, can be injected into the animal. The test somatostatin analog is then injected into the animals at various concentrations. Blood samples can be taken from the animal, and the amount of amylin present before and after the injection of the test somatostatin analog can be determined by radioimmunoassay.
By xe2x80x9camylin release stimulatorxe2x80x9d is meant a compound which stimulates the release of amylin stored in pancreatic cells. Examples of amylin release stimulators include glucose, D-glyceraldehyde, or L-arginine.
The method described above can be used to screen for new compounds capable of inhibiting amylin release from the pancreas in a patient. To promote efficiency, when the method is used in a screening project, two or more test compounds can be linked together as a single sample, and, if necessary, subsequently divided and retested.
Another aspect of this invention relates to a method of treating (i.e., either preventing or ameliorating) hyperamylinemia by inhibiting release of amylin from the pancreas in a subject (i.e., a mammal, such as a human being) who suffers from, or is predisposed to suffer from, hyperamylinemia. The method includes administering (e.g., parenterally, intravenously, subcutaneously, transdermally, intramucously, or via implantation of a sustained release formulation), to the subject an amount of a SSTR-5 agonist, the amount being effective in treating hyperamylinemia, i.e., in lowering amylin levels in the bloodstream by inhibiting the release of amylin from pancreas cells.
What is meant by xe2x80x9cSSTR-5 agonistxe2x80x9d is a compound which (i) is more selective for SSTR-5 than for SSTR-2, i.e., its Ki for SSTR-5 is lower than that for SSTR-2 (as determined by one of the receptor binding assays described in the working examples below); and (ii) inhibits the release of amylin from pancreas cells induced by an amylin release stimulator (as determined by one of the functional assays described in the working examples below). Both examples of SSTR-5 agonists and the procedures of selecting them appear in the xe2x80x9cDescription of the Inventionxe2x80x9d section below.
The above-described therapeutic method can be used to treat a subject suffering from pancreatic cancer, prediabetic symptoms, or non-insulin dependent diabetics.
An effective amount depends upon the conditions being treated, the route of administration chosen, and the specific activity of the compound used, and ultimately will be decided by the attending physician or veterinarian. While it is possible for a SSTR-5 agonist to be administered as the pure or substantially pure compound, it is preferable to present it as a pharmaceutical formulation or preparation. The formulations to be used in the present invention, for both humans and animals, comprise any of the SSTR-5 agonists to be described below, together with one or more pharmaceutically acceptable carriers and optionally other therapeutic ingredients. The carrier must be xe2x80x9cacceptablexe2x80x9d in the sense of being compatible with the active ingredient(s) of the formulation (and preferably, capable of stabilizing peptides) and not deleterious to the subject to be treated.
The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing the active ingredient(s) into association with the carrier which constitutes one or more accessory ingredients.
In general, the formulations for tablets or powders are prepared by uniformly and intimately blending the active ingredient with finely divided solid carriers, and then, if necessary as in the case of tablets, forming the product into the desired shape and size.
Formulations suitable for intravenous or subcutaneous administration, on the other hand, conveniently comprise sterile aqueous solutions of the active ingredient(s). Preferably, the solutions are isotonic with the blood of the subject to be treated. Such formulations may be conveniently prepared by dissolving solid active ingredient(s) in water to produce an aqueous solution, and rendering the solution sterile. The formulation may be presented in unit or multi-dose containers, for example, sealed ampoules or vials. Delivery of drug by parenteral implantation of a sustained release formulation is well-known in the art. See, e.g., U.S. Pat. Nos. 3,773,919, 4,767,628; and PCT Application No. WO 94/00148.
Also within the scope of this invention are a SSTR-5 agonist for use in treating a disease or a disorder relating to hyperamylinemia, and the use of a SSTR-5 agonist for the manufacture of a medicament for the treatment of such disease or disorder.
Use of a ligand more selective for SSTR-5 than for SSTR-2 to treat hyperamylinemia minimizes undesirable side effects.
Other features and advantages of the invention will be apparent from the following drawings and detailed description of several embodiments and from the appending claims.